Method Optimisation in Bottom-Up Analysis of Proteins

Transcription

1 Method Optimisation in Bottom-Up Analysis of Proteins M. Styles, 1 D. Smith, 2 J. Griffiths, 2 L. Pereira, 3 T. Edge 3 1 University of Manchester, UK; 2 Patterson Institute, Manchester, UK; 3 Thermo Fisher Scientific, Runcorn, Cheshire, UK

2 Overview Purpose: This poster will provide guidance information on how to optimize the analytical procedure for analyzing proteins using a bottom up approach, both in terms of the sample preparation and also the subsequent peptide separation. Methods: An investigation is performed to look at approaches to optimizing the five individual steps in generation of a series of peptides from a protein; 2 Method Optimisation in Bottom-Up Analysis of Proteins Denaturing the protein Reduction of disulfide bonds Alkylation Desalting Digestion Finally, the parameters that affect the chromatography, including the use of ion pairing reagents, ph and methods to improve the peak capacity will be discussed. Results: The results demonstrate the importance of optimizing each of the sample preparation steps and also the importance of having high resolution chromatography for the final analysis. Introduction Optimisation of the sample preparation and chromatography conditions when performing a bottom up analysis of a protein mixture ensures that a better detection of peptides occurs which ultimately allows better identification of the parent protein molecules. Initially a BSA protein was used to demonstrate the importance of optimizing the sample preparation, with YLYEIAR, being the peptide that was monitored. For the sample preparation, five key stages exist; Denaturing the protein Denaturing the protein allows the protease enzyme to access the whole protein backbone resulting in better peptide recovery, and higher sequence coverage upon analysis. Reduction of disulfide bonds Dithiothreitol (DTT) reduces the disulfide bonds between cysteine residues without affecting other amino acids in the protein. This allows the protein to become more fully unfolded. Alkylation An alkylating agent such as iodoacetic acid is added to alkylate all of the cysteine residues preventing the formation of disulfide bonds, so that the protein remains unfolded. Desalting Salts and other reagents that may denature the enzyme need to be removed or diluted to ensure successful digestion. In all cases SPE was used to desalt the sample. Digestion The enzyme (trypsin) is added to the resulting solution with a 1:20 mass ratio to the protein in the solution. Trypsin activity is highest between ph 8-9 and hence the solution is generally buffered to this ph range with Tris or ammonium bicarbonate. Other enzymes can be used but they are less specific.

3 Methods The starting method was as follows, with the parameters that were altered highlighted in the relevant section. Denaturing: A solution containing 5 mg/ml acetylated BSA was made up in 100 mm ammonium bicarbonate containing 6 M guanidine HCl as a denaturant. The 200 μl aliquot was left to denature for 30 minutes at 37 C. Reduction: achieved by the addition of 5 μl of DTT stock solution, vortexed, and left for 30 minutes at 37 C. Alkylation: carried out by addition of 4 μl of iodoacetic acid stock solution for alkylation, vortexed and left for 30 minutes at 37 C. Alkylation was then stopped by addition of 20 μl of DTT stock solution to quench the reaction. Desalting: The solution was then diluted with 800 μl of the ammonium bicarbonate buffer to reduce the concentration of the chaotropic agent (guanidine HCl) to below 1 M. Digestion: 20 μg of lyophilised trypsin was reconstituted and activated in 20 μl of 0.01% TFA. This solution was diluted down so that the enzyme concentration was reduced 20 μg/ml by addition of 980 μl of ammonium bicarbonate buffer μl of the trypsin solution was added to the sample, at a ratio of 1:50 (m/m) and given a gentle shake to aid reconstitution. The sample was left to digest over night at 37 C. Desalting: The resulting digests were acidified by addition of 20 μl of 10% formic acid. The samples were applied to the 3 ml HyperSep C18 100mg cartridges for desalting, and forced through manually using positive pressure. The loaded cartridge was washed with 3 ml of 0.1% formic acid to remove the salts and the sample was then eluted with 400 μl 50/50/0.1 acetonitrile/water/formic acid (v/v/v). The peptides of interest all elute in lower organic concentrations than this. The extracts were then blown down to dryness and reconstituted in the starting mobile phase. Thermo Scientific Poster Note PN20819_e 06/13S 3

4 Results Step 1. Denaturing the Protein Figure 1 demonstrates a chromatogram of two peptides and the amount of each produced from a digestion procedure where the only variable is the type of denaturing reagent used, either urea or guanidine HCl. It can be seen from this figure that the reagent has a significant effect on the abundance of individual peptides being observed. FIGURE 1. Effect of varying the denaturing reagent. The next set of experimental data obtained is shown in Figure 2, and looks at the effect of varying the reaction time and the reaction temperature on the denaturing step. It can be seen that in general increasing the temperature and increasing the reaction time increases the yield. FIGURE 2. Effect of varying the temperature and duration of the denaturing step. Step Alkylation and Reduction of the Protein Figure 3 demonstrates the effect that temperature and time have on the recovery of a single peptide in the alkylation and reduction steps. In this example it can be seen that the hotter the reaction conditions the worse the recovery. The duration of the reduction time does not appear to have a significant effect. The alkylating reagent used in this case was DTTP. 4 Method Optimisation in Bottom-Up Analysis of Proteins

5 FIGURE 3. Effect of varying duration of the reduction step and the temperature of the alkylation step. Step 4. Desalting the Protein There is little to be optimized on this step, and so it was not investigated further. Step 5. Digestion of the Protein Figure 4 demonstrates the effect of changing the duration and concentration of enzyme added. It can be seen that both the duration and the enzyme concentration increase the amount of peptide measured. FIGURE 4. Effect of varying parameters in the digestion step. Solution A - Control Solution B- double enzyme concentration Solution C - digestion time 60 hours A B C Thermo Scientific Poster Note PN20819_e 06/13S 5

6 Chromatography Development Figure 5 highlights some challenges associated with the development of peptide chromatography. It can be seen that the peak shape is improved when using TFA compared to using formic acid, however there is also a loss of detector sensitivity for the peptide as the concentration of TFA is increased. The ph of the mobile phase can also have an effect on the overall charge state of the protein and this in turn will have a effect on the retention, Figure 6. FIGURE 5. Effect of the addition of differing acids to sensitivity and peak shape. FIGURE 6. Effect of the ph on the retention of peptides. ph 9.6 TVMENFVAFVDK RHPEYAVSVLLR AEFVEVTK LVTDLTK ph 1.9 TVMENFVAFVDK AEFVEVTK RHPEYAVSVLLR LVTDLTK Two Dimensional Chromatography To improve the peak capacity a two dimensional approach was employed, Figure 7. This used a combination of fraction collection from the 1 st dimension and a further separation on the second dimension. Two modes of initial fractionation were considered either SCX or the use of PGC, Figure 8. It can be clearly seen that the use of PGC enhances the peptide sequence and hence protein determination, since the peptides are more evenly detected in the fractions collected. 6 Method Optimisation in Bottom-Up Analysis of Proteins

7 FIGURE 7. Schematic diagram of 2D fraction collection experiment. 96% ACN 0.1% TFA 0.25 ml min -1 1 st Dimension 400 µg Sample 25% 2% 40 min PGC 2.1 mm x 100mm x 5 µm Hypercarb TM UV signal at 254 nm 2 nd Dimension ACN 0.1% FA 0.25 ul min -1 3% 30 min C18 75 um x 250mm <2.0 um 4% injected TIC from each fraction FIGURE 8. Comparison of number of peptides identified using SCX and PGC. Conclusion The optimization of the sample preparation procedure can improve the sensitivity of the assay by a factor of 5 compared to non-optimized conditions with the denaturing step being the most sensitive. Optimization of the chromatography can be achieved using careful selection of ph and also using complementary 2D HPLC Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Australia Austria Belgium Brazil China Denmark France Germany India Italy Japan Korea Netherlands Singapore Sweden Switzerland Taiwan UK/Ireland USA and Canada PN20819_E 07/16S